tailieunhanh - Báo cáo khoa học: Pressure and heat inactivation of recombinant human acetylcholinesterase Importance of residue E202 for enzyme stability

The effects of pressure on structure and activity of recom-binant human acetylcholinesterase (rHuAChE) were inves-tigatedup toapressureof 300 MPausinggel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity meas-urements followingexposure tohighpressure. | Eur. J. Biochem. 269 4297-4307 2002 FEBS 2002 doi Pressure and heat inactivation of recombinant human acetylcholinesterase Importance of residue E202 for enzyme stability Cecile Clery-Barraud1 Arie Ordentlich2 Haim Grosfeld2 Avigdor Shafferman2 and Patrick Masson1 1 Centre de Recherches du Service de Sante des Armees Unite d enzymologie France 2Israel Institute for Biological Research Department of Biochemistry and Molecular Biology Ness-Ziona Israel The effects of pressure on structure and activity of recombinant human acetylcholinesterase rHuAChE were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure fluorescence of bound 8-anilinonaphthalene-1-sulfonate ANS and activity measurements following exposure to hihh pressuee. Study of wdd-type enzyme and three single mutants D74N E202Q E450A and one sextuple mutant E84Q E292A D349N E358Q E389Q D390N showed that pressure exerts a differential action on wild-type rHuAChE and its mutants allowing estimation of the contribution of Oilrboxy 1 ic 0 acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE. Keywords pressure inactivation protein stability acetylcholinesterase mutants. Acetylcholinesterase AChE EC plays a central role in the cholinergic system by rapidly hydrolyzing the neurotransmitter acetylcholine. Organophosphorus compounds OPs pesticides insecticides drugs and chemical warfare agents nerve gases inhibit cholinesterases ChEs by phosphylatingtheir active-site serine. .